Influence of ß-lactam antibiotics on the activity of K. pneumoniae bacteria biofilm forms isolated from wounds of patients with chronic osteomyelitis.

One of the reasons for the emergence of highly resistant strains is associated with the ability of bacteria to form biofilms on various surfaces. The formation of a biofilm by pathogens leads to a decrease in the activity of the antibiotic, an increase in the time for the production of stress response genes by bacteria, and, as a result, an increase in antimicrobial tolerance. To investigate the effect of imipenem and cefepime on the activity of biofilm forms of K. pneumoniae bacteria isolated from the wounds of patients with chronic osteomyelitis. The object of the study is clinical strains of K. pneumoniae isolated from the wounds of patients with chronic osteomyelitis. In the control series, the level of biofilm formation of K. pneumoniae strains was assessed after 48 hours of cultivation on coverslips and 96-well polystyrene plates. In the second and third series, the biofilm form of K. pneumoniae bacteria was exposed to imipenem and cefepime, and after 24 hours the activity of biofilm formation was assessed according to previously developed criteria. The structure of the emerging biofilm on the surface of the coverslip in all series of the experiment was represented by single adherent cells and microcolonies of various sizes. Cultivation with antibiotics led to a decrease in the number of microcolonies ranging in size from 10 to 10,000 µm2 in the second and third series, however, significant differences from the control series were found only when exposed to cefepime. The intensity of film formation of K. pneumoniae in the control series by the tablet method was 0.350 (0.334; 0.368) units opt.pl. When cultivating biofilms together with antibacterial drugs, the biofilm-forming activity after 24 hours of the experiment was significantly lower than in the control group in all experimental series. K. pneumoniae bacteria isolated from patients with chronic osteomyelitis, when cultivated on polystyrene plates and on the surface of coverslips, actively form a biofilm, exhibiting highly adhesive properties. The studied antibiotics were shown to have a bacteriostatic effect on biofilm forms of K. pneumoniae bacteria. The bactericidal effect of imipenem and cefepime on biofilm forms was not revealed.

Efficacy of cephalosporins against enterobacteria isolated from patients with chronic osteomyelitis.

Long-term antibiotic therapy, as well as inappropriate use of drugs in the treatment of osteomyelitis, can lead to the appearance of pan-resistant strains. The existing antibiotic prophylaxis regimens for purulent-septic complications are outdated and need to be adjusted. In this regard, it is necessary to monitor the resistance of microorganisms in order to identify ineffective antibacterial drugs. To analyze the resistance profiles of Enterobacteriaceae isolated from patients with chronic osteomyelitis to cephalosporin drugs over a three-year period. The resistance profiles of 912 clinical strains of Enterobacteriaceae were analyzed: Klebsiella pneumoniae (n=349), Proteus sp. (n=208), Escherichia coli (n=176), Enterobacter cloacae (n=179) for the period from 2018-2020 to cephalosporin drugs. In 2018, 66.2% of Enterobacteriaceace were resistant to the 1st generation cephalosporins, in 2019 - 78.7%, in 2020 - 79.5%. Generation II cephalosporins were most active against Proteus sp. bacteria, but a decrease in clinical effect was observed by 2020. Among the third generation cephalosporins in 2018, cefotaxime was most active, but in 2020 the number of resistant strains doubled and amounted to 86.3%. Ceftazidime was active against 47.1% of Enterobacteriaceae isolates in 2018, in 2019 - 45% of strains, in 2020 - 37.2% of bacterial strains. High activity of ceftriaxone was noted only in 2018 against Proteus sp. Preparations of the IV generation in 2018 showed the highest activity against bacteria of the genus Proteus, the least - against bacteria K. pneumoniae. In the period from 2019-2020, a significant decrease in the effectiveness of cefepime was observed.The monitoring of the resistance profiles to antibiotics of the cephalosporin series revealed their low efficacy against Enterobacteriaceae isolated from wounds and fistulas of patients with chronic osteomyelitis, which shows the inexpediency of their empirical use.

Monitoring of the leading causative agents of osteomyelitis and their antibiotic resistance.

The annual monitoring of the species composition of the causative agents of osteomyelitis, the identification of antibiotic-resistant strains, the study of the species composition of associations of microorganisms, their adhesive activity will prevent the spread of infection. Analyze the spectrum of the leading causative agents of osteomyelitis, their antibiotic sensitivity, and also the adhesive activity of the identified bacterial associations. A microbiological analysis of 2197 smears of adult patients with various etiological forms of osteomyelitis who were treated in the departments of the purulent center of the FSBI «NMRCTO¼ of the RF Ministry of Health in 2019. The spectrum of pathogenic microflora, sensitivity to standard antibacterial drugs used in the clinic was studied. The biofilm-forming ability of associations of microorganisms was investigated. According to the conducted microbiological monitoring for 2019, the microflora spectrum for osteomyelitis is diverse, the main pathogens are S. aureus, S. epidermidis, P. aeruginosa, K. pneumoniae, Enterococcus sp. A high percentage of isolation of microbial associations was noted, most often mix cultures of gram-positive and gram-negative bacteria. Bacterial associations: S. aureus + P. aeruginosa, S. aureus + S. marcescens, S. aureus + A. baumannii, S. epidermidis + E. cloacae - actively formed a biofilm on the surface of polystyrene plates, and the adhesive potential depended on interstrain relations in the composition of the formed biofilm. Among Gram-negative microflora, multiresistant strains prevail, among Gram-positive microflora - a high percentage of methicillin-resistant Staphylococci. When analyzing the antibiotic sensitivity of the isolated microorganisms, a high percentage of resistant strains is noted. So, with respect to enterobacteria, ß-lactam antibiotics, drugs from the group of aminoglycosides, turned out to be ineffective. Among non-fermenting gram-negative bacteria, A. baumannii strains had multiple antimicrobial resistance. Among gram-positive microorganisms, a high percentage of isolation of methicillin-resistant staphylococci was noted. The specificity of the course of the disease and measures aimed at eliminating the pathogen depend on the species composition in the focus of infection. The study of the etiological structure of osteomyelitis, the monitoring of the antibiotic resistance of pathogens and their persistent potential, makes it possible to adopt sound tactics of conservative and surgical treatment.

[Dependence of biofilm-forming ability on the antibiotic sensitivity of Pseudomonas aeruginosa clinical strains isolated from patients with chronic osteomyelitis.]

Due to the spreading highly resistant strains among clinically significant P. aeruginosa clones, it becomes necessary to prescribe antibiotics not only taking into account the knowledge of sensitivity spectrum of a particular isolate but the data of microorganism biofilm activity as well. To study the dependence of biofilm-forming ability on the sensitivity to antibacterial preparations of P. aeruginosa clinical strains, isolated from patients with chronic osteomyelitis. 36 patients above 18 with chronic osteomyelitis of long tubular bones who were treated in the center of purulent osteology took part in the experiment. Object of the study - material isolated from wounds, fistulas, as well as from inflammatory foci. The sensitivity of isolated microorganisms to 10 antibiotics was analyzed: Piperacillin/Tazobactam, Imipenem, Meropenem, Aztreonam, Amikacin, Ciprofloxacin, Ceftriaxone, Ceftazidime, Cefotaxime, Cefepime. High- and medium-adhesive strains accounted for 86,1 % among P. aeruginosa clinical isolates, obtained from the wounds of patients with chronic osteomyelitis of long tubular bones. Highly adhesive strains are resistant to a wide range of antibacterial preparations used clinically. Penicillins were the most effective preparations when analyzing antibioticograms obtained for highly adhesive strains, for medium adhesive strains - penicillins, aminoglycosides and carbapenems, for low adhesive ones - aminoglycosides, penicillins, carbapenems, monobactams, quinolones. P. aeruginosa multi-resistance is a serious problem in the treatment of patients with chronic osteomyelitis. Spreading antibiotic-resistant strains of P. aeruginosa is associated with the presence of bacteria in the biofilm. Since adhesion is the first step in the biofilm formation, it is important to identify strains having high adhesive ability timely.

[Microflora of surgical wounds and fistulas in patients with chronic osteomyelitis of the tibia before reconstructive treatment, in case of recurrence of infection.]

Relevance of microbiological monitoring in chronic osteomyelitis of the tibia developed during treatment of fractures with a plate is associated with a noticeable increase of various kinds of the microflora. A microbiological study was conducted of pathological material taken from wounds, fistulas and from the focus of inflammation in 49 patients with chronic tibial osteomyelitis, developed during treatment of fractures with a plate. The patients underwent sequestrectomy of the tibia and subsequent application of bilocal consecutive compression-distraction osteosynthesis or monolocal compression osteosynthesis. Microbiological study of smears taken before the reconstructive treatment from fistulas and wounds of patients showed that in monoculture there were 30 strains, the remaining 30 - as a part of 14 two - and three-component associations. The frequency of S. aureus strains was 55.3%, followed by coagulase-negative staphylococcus - 13.6% and representatives of the family Enterobacteriacae - 10.2%. There were methicillin-resistant strains of S. aureus in 11.8%, strains of coagulase-negative staphylococcus (MRCoNS) - 6.8%. Recurrence of the disease was observed in 7 patients. The microflora of the discharge from the fistula was represented by monocultures of S. aureus and associations of bacteria in which one of the components was methicillin-resistant strains of S. aureus. We observed differences in the contents of the microflora before reconstructive treatment of patients and in recurrence of infection. In case of recurrence of infection, the qualitative contents of the microflora changed: in 2 patients in the association of microorganisms and in 3 - in monocultures, S. aureus strains acquired resistance to ß-lactam antibiotics, new associations appeared, which were not present in primary cultures before reconstructive treatment. The study showed that the spectrum of micro-organisms in chronic osteomyelitis of the tibia, developed after osteosynthesis with a plate, varied and is subject to change and that dictates the need for microbiological monitoring to detect the etiological structure of pathogens, monitoring of antibiotic resistance of the isolated strains and rational approach to treatment of patients.

[THE BIOFILM FORMATION ABILITY OF STRAINS SERRATIA SPP., SEPARATED FROM WOUNDS OF PATIENTS WITH CHRONIC OSTEOMYELITIS IN MONO-CULTURES AND IN COMPOSITION OF ASSOCIATION OF MICROORGANISMS HARVESTED IN VITRO].

The article presents analysis of characteristics of 7 clinical strains of Serratia marcescens separated from fistulas in pre-operational period and from nidus of inflammation during operations in 2013-2015. in 7 patients with chronic osteomyelitis of long tubular bones. The bacteria S.marcescens are separated in one patient in monoculture and in 6 patients in composition of associations: S.marcescens + S.warneri + P.aeruginosa (n=1); S.marcescens + S.aureus (n=4); S.marcescens + M.morganii (n=1). According phometric analysis, strains of S.marcescens were characterized by average ability for biofilm formation on the surfaces of polystyrene dishes and cover glass that is substantiated by values of optic density and is conformed to data of their adhesion activity. The biofilm formation ability of association of microorganisms (S.marcescens + P.aeruginosa, S.marcescens + M.morgani) already after 24 hours of experiment were correspondingly higher in 1.4 and 1.2 times of levels of biofilm formation in monocultures. The activity of biofilm formation of association (S.marcescens + S.aureus) on the first day of experiment was low. However, after 48 hours a significant growth of biofilm was observed that is substantiated by values of optical density. The results of study demonstrated ability of all clinical strains of S.marcescens separated from osteomyelitis nidus to adhesion on surface of erythrocytes and to biofilm formation on abiotic surfaces (polystyrene and glass) both in monoculture and associations with other microorganisms. In consideration that microbial films play leading role in chronization of infectious diseases it is necessary to be aware of seriousness of etiologic role of S.marcescens in development of osteomyelitis as a pathogen.

[The clinical significance of bacteria Acinetobacter spp., separated from patients with chronic osteomyelitis.]

The article deals with the results of analysis of 17 clinical strains of Acinetobacter spp., isolated from wounds of patients with chronic osteomyelitis of long bones. In 8 patients strains of Acinetobacter spp. were isolated in mono-culture, in 8 - in composition of association with staphylococci (Staphylococcus aureus - 4, S.epidermidis - 2, S.hatmolyticus - 1, S.capitis -1) and in one patient - with strains of Enteroccocus faecalis. highly adhesive characteristics were established in 29% of isolates of Acinetobacter spp, average adhesive - in 43% and low adhesive - in 28%. The average index of adhesiveness of analyzed strains made up to 2.86±0.02 units. The strains with high adhesive potential were isolated from associations with Staphylococcus spp. The strains of Acinebacter spp. are characterized as far as of incubating by increasing activity of formation of biofilm on the surface of 96 alveolar tray. The calculated integral coefficient (K>0.5 units) testifies high percentage of strains resistant to selected antibiotics. It is established that the most effective in relation to Acinebacter spp. were aminoglycosides (gentamycin and tobramycin) and carbapenems (meropenem, imipenem).The percentage of resistant strains did not exceed 38%. The implemented study demonstrated that inter-microbial relationships ameliorate capacity of association-forming strains Acinebacter spp. to form bio-films. The derived values of bio-film-forming capacity and coefficient of resistance of clinical isolates Acinebacter spp.isolated from patients with chronic osteomyelitis testify their high pathogenicity. The treatment of infection associated with Acinebacter spp. is to consider both results of antibioticgram and data concerning virulent characteristics of isolated agent.

[The evaluation of adhesive activity of bacteria extracted from patients with infected endoprostheses of large joints].

The article presents the results of study of adhesive characteristics of 67 clinical strains of bacteria extracted from patients with infected endoprotheses of large joints. All analyzed strains of microorganisms had adhesive characteristics. The unequal distribution by degree of adhesiveness for analyzed groups is marked. The capability to adhesiveness in Gram-negative microorganisms is higher in comparison with Gram-positive microorganisms. The Gram-negative microorganisms form biofilms more actively.